Elevated levels of homocysteine constitute a significant, independent, and graded risk factor for cardiovascular diseases. In addition, high plasma homocysteine is correlated with the occurrence of neural tube defects, the most common type of birth defect, and with Alzheimer's disease. Homocysteine is a junction metabolite that can either be salvaged back to the methionine cycle via the action of transmethylases or be committed to cysteine biosynthesis via the transsulfuration pathway. The reaction catalyzed by cystathionine beta-synthase, an enzyme that is unique in its dependence on heme and pyridoxal phosphate (PLP) for activity, represents the first of two steps that convert homocysteine to cysteine. Mutations in cystathionine beta-synthase are the single most common cause of hereditary hyperhomocysteinemia, characterized by catastrophically high levels of plasma homocysteine with attendant early and aggressive occlusive arterial diseases. In this study, we propose to examine the role of the two cofactors, heme and PLP, and of the allosteric effector, S- adenosylmethionine, in the cystathionine beta-synthase-catalyzed reaction. While the beta replacement chemistry catalyzed by cystathionine beta-synthase suggests an obvious role for PLP, the role of the heme is enigmatic. Studies from our laboratory suggest a regulatory sensor role for the heme. Experiments designed to address the following questions are described in this proposal. (i) What are the intermediates in the reaction cycle catalyzed by cystathionine beta-synthase? (ii) How is the activity of the enzyme regulated by heme and by the allosteric effector, S-adenosylmethionine? (iii) What are the catalytic penalties associated with a select number of missense mutations identified in cystathionine beta-synthase-deficient hyperhomocysteinemic patients? (iv) Does human cystathionine beta-synthase interact with other proteins that modulate its function in the cellular milieu? A combination of biophysical techniques including rapid-reaction kinetics and EPR spectroscopy, and cell biological methods including fluorescence immunolocalization of cystathionine beta-synthase in brain tissue and immunoprecipitation methods will be employed to address the questions that are being posed. These studies will fill significant gaps in our understanding of this key enzyme and advance the field of homocysteine biology.